Interferon gamma (IFNgamma) has been shown to inhibit proliferation and differentiation of erythroid progenitor cells and to produce apoptosis of erythroid cells, whereas stem cell factor (SCF), erythropoietin (EP), and insulin-like growth factor-I (IGF-I) have distinct roles in enhancing erythroid cell production and preventing apoptosis. The mechanism by which IFNgamma exerts an inhibitory effect on the positive roles of these growth factors is unknown. Although some inhibitory cytokines including IFNgamma have been shown to downregulate growth factor receptors, the effect of IFNgamma on SCF, EP, and IGF-I receptors of human erythroid progenitor cells has not been defined. We obtained highly purified day-5 or day-6 erythroid colony-forming cells (ECFCs) from human blood in sufficient quantity and purity for radiolabeled cytokine binding studies and analysis of mRNA. When day-5 ECFCs were incubated with increasing concentrations of recombinant human (rh) IFNgamma for 24 hours at 37 degrees C, specific binding of 125I-rhSCF to SCF receptors was significantly decreased by 25% to 40% in a dose-dependent fashion, with the maximum effect at 2,500 to 5,000 U/mL of IFNgamma. The decrease was apparent by 12 hours of incubation and was only slightly lower by 24 hours. The numbers of SCF and EP receptors, but not of IGF-I receptors, per ECFC, calculated by Scatchard analysis, were significantly decreased by 30% and 23% to 25%, respectively, after incubation with 2,500 U/mL rhIFNgamma for 24 hours at 37 degrees C, whereas the binding affinities were not affected. This decrease in SCF receptors was confirmed by flow cytometry using an anti-c-kit mouse monoclonal antibody. Northern blot analysis showed that the mRNAs for the SCF and EP receptors, but not for the IGF-I receptors, were decreased by 50% to 60% after 3 hours of incubation at 37 degrees C with 2,500 U/mL of rhIFNgamma. This persisted for 24 hours without alteration of the stability of the SCF and EP receptor mRNAs. These observations suggest that one means by which IFNgamma inhibits erythroid cell proliferation and differentiation and produces apoptosis may be through the reduction of the number of target receptors for SCF and EP and that this occurs through transcriptional inhibition of the corresponding mRNAs.